Functional Investigation of the Novel BRCA1variant (Glu1661Gly) byComputationalTools andYeastTranscription Activation Assay
Publish place: Multidisciplinary Cancer Investigation، Vol: 3، Issue: 2
Publish Year: 1398
نوع سند: مقاله ژورنالی
زبان: English
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شناسه ملی سند علمی:
JR_MCIJO-3-2_003
تاریخ نمایه سازی: 15 بهمن 1399
Abstract:
Introduction: Mutations in the BRCA1 gene are major risk factors for breast and ovarian cancers. However, the relationship between some BRCA1 mutations and cancer risk remains largely unknown. Cancer risk predictions could be improved by evaluation of the impairment degree in the BRCA1 functions due to a specific mutation. This study aimed to assess the functional effect of a novel variant (Glu1661Gly) by a combination of in silico tools, structural analysis, and also experimental functional assay based on yeast transcription activation.
Methods: Computational tools including PROVEAN, PolyPhen2, Align-GVGD, Mutation Taster, and also structural analysis were used for prediction of the impact of Glu1661Gly on protein function. To perform the yeast functional assay, the BRCA1 C-terminal (BRCT domain) was cloned into pLexA plasmid in-frame with the DNA-binding domain of LexA to generate a functional transcription activator. The resulted construct was transformed into EGY48/ pRB1840 yeast and positive colonies were assayed for β-galactosidase activity. Wild-type BRCA1 and Ser1613Gly were used as positive controls and Met1775Arg as negative control.
Results: The Glu1661Gly variant was predicted to be neutral by PROVEAN, disease- causing by Mutation Taster, probably damaging by Polyphen2, and intermediate effect by Align-GVGD. The yeast functional assay revealed that Glu1661Gly activity was comparable to wild-type BRCA1
Conclusions: Observed discrepancies between in silico tools make it difficult to interpret the results. Based on structural analysis, the Glu1661Gly on α1 helix of the C-terminal domain does not seem to impair function due to α1 helix is far from the BRCT-BRCT interface and phosphopeptide-binding site. This variant was also classified as neutral; using yeast functional assay.
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Authors
Fatemeh Yadegari
Genetics Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
Leila Farahmand
۲ Recombinant Proteins Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
Rezvan Esmaeili
Genetics Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
Shiva Zarinfam
Genetics Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
Keivan Majidzadeh-A
Genetics Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran
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